Modified adeno-associated virus vector compositions

ABSTRACT

An adeno-associated virus filler component comprising a nucleic acid of between 3300 and 4200 nucleotides in length is disclosed.

RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.15/363,182, filed on Nov. 29, 2016, which is a continuation of U.S.application Ser. No. 14/410,983, filed on Dec. 23, 2014, which is aNational Stage application under 35 U.S.C. §371 of InternationalApplication No. PCT/US2013/031644, having an international filing dateof Mar. 14, 2013, which claims the benefit of priority of U.S.Application Ser. No. 61/668,839, filed Jul. 6, 2012, which applicationsare incorporated by reference herein.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 14, 2013, isnamed 17023_126 WO1_SL.txt and is 39,125 bytes in size.

BACKGROUND

Adeno associated virus (AAV) is a small nonpathogenic virus of theparvoviridae family. AAV is distinct from the other members of thisfamily by its dependence upon a helper virus for replication. Theapproximately 5 kb genome of AAV consists of one segment of singlestranded DNA of either plus or minus polarity. The ends of the genomeare short inverted terminal repeats which can fold into hairpinstructures and serve as the origin of viral DNA replication. Physically,the parvovirus virion is non-enveloped and its icosohedral capsid isapproximately 20 nm in diameter.

To-date many serologically distinct AAVs have been identified and havebeen isolated from humans or primates. Govindasamy et al., “StructurallyMapping the Diverse Phenotype of Adeno-Associated Virus Serotype 4,” J.Vir., 80 (23):11556-11570 (2006). For example, the genome of AAV2 is4680 nucleotides in length and contains two open reading frames (ORFs).The left ORF encodes the non-structural Rep proteins, Rep 40, Rep 52,Rep 68 and Rep 78, which are involved in regulation of replication andtranscription in addition to the production of single-stranded progenygenomes. Rep68/78 has also been shown to possess NTP binding activity aswell as DNA and RNA helicase activities. The Rep proteins possess anuclear localization signal as well as several potential phosphorylationsites. Mutation of one of these kinase sites resulted in a loss ofreplication activity.

The ends of the genome are short inverted terminal repeats (ITR) whichhave the potential to fold into T-shaped hairpin structures that serveas the origin of viral DNA replication. Within the ITR region twoelements have been described which are central to the function of theITR, a GAGC repeat motif and the terminal resolution site (trs). Therepeat motif has been shown to bind Rep when the ITR is in either alinear or hairpin conformation. This binding serves to position Rep68/78for cleavage at the trs which occurs in a site- and strand-specificmanner.

The following features of AAV have made it an attractive vector for genetransfer. AAV vectors possess a broad host range; transduce bothdividing and non-dividing cells in vitro and in vivo and maintain highlevels of expression of the transduced genes. Viral particles are heatstable, resistant to solvents, detergents, changes in pH, temperature,and can be concentrated on CsCl gradients. AAV is not associated withany pathogenic event, and transduction with AAV vectors has not beenfound to induce any lasting negative effects on cell growth ordifferentiation. The ITRs have been shown to be the only cis elementsrequired for packaging allowing for complete gutting of viral genes tocreate vector systems.

There is a current need for AAV vectors that have improved packagingfeatures.

SUMMARY

In certain embodiments, the present invention provides anadeno-associated virus (AAV) filler component (also called a “stuffersequence”) comprising a nucleic acid of between 3300 and 4200nucleotides in length having at least 90% identity to SEQ ID NO:1 or SEQID NO:2.

In certain embodiments, the present invention provides anadeno-associated virus (AAV) filler component consisting of a nucleicacid of between 3300 and 4200 nucleotides in length having at least 90%identity to SEQ ID NO:1 or SEQ ID NO:2.

In certain embodiments, the present invention provides an AAV vectorcomprising the filler component described above.

BRIEF DESCRIPTION OF THE DRAWINGS AND TABLE

FIG. 1 is a plasmid map of 5pFBAAVmU6miHDS1stuffer (9110 bp).

FIGS. 2A-2R provide the sequence of 5pFBAAVmU6miHDS1stuffer (Stuffer #1)(SEQ ID NO:3).

FIGS. 3A-3C provide the sequences of the various individual componentsof 5pFBAAVmU6miHDS1stuffer (SEQ ID NO:1, 4-11).

FIG. 4 is a graph showing relative Htt expression.

FIG. 5 is a plasmid map of 5pFBAAVmU6miHDS1-stuffer.

FIGS. 6A-6D provide the plasmid sequence for 5pFBAAVmU6miHDS1-stuffer(SEQ ID NO:12).

FIGS. 7A-7B provide a stuffer sequence (Stuffer #2) (SEQ ID NO:2).

FIG. 8. EM evaluation of full virions vs. empty virions. Two examples ofempty virions are highlighted by the arrows. This prep had only ˜4%empty virions, which is quite low.

FIG. 9. Silver stain to examine the capsid integrity of the purifiedvirions. Several different miRNA-expressing constructs were engineeredinto the shuttle vector along with the intron I/II stuffer to generatenear wild type genome size. The purified viruses show optimal VP1, VP2and VP3 protein ratios.

Table 1. % Packaging efficiencies of miR-intronI/II virions and %contaminants.

DETAILED DESCRIPTION

AAV Vectors and Expression Cassettes

The viral vectors of the invention utilize an AAV vector. An “AAV”vector refers to an adeno-associated virus, and may be used to refer tothe naturally occurring wild-type virus itself or derivatives thereof.The term covers all subtypes, serotypes and pseudotypes, and bothnaturally occurring and recombinant forms, except where requiredotherwise. As used herein, the term “serotype” refers to an AAV which isidentified by and distinguished from other AAVs based on capsid proteinreactivity with defined antisera, e.g., there are eight known serotypesof primate AAVs, AAV-1 to AAV-8. For example, serotype AAV-2 is used torefer to an AAV which contains capsid proteins encoded from the cap geneof AAV-2 and a genome containing 5′ and 3′ ITR sequences from the sameAAV-2 serotype.

Pseudotyped AAV refers to an AAV that contains capsid proteins from oneserotype and a viral genome including 5′-3′ ITRs of a second serotype.Pseudotyped rAAV would be expected to have cell surface bindingproperties of the capsid serotype and genetic properties consistent withthe ITR serotype. Pseudotyped rAAV are produced using standardtechniques described in the art. As used herein, for example, rAAV1 maybe used to refer an AAV having both capsid proteins and 5′-3′ ITRs fromthe same serotype or it may refer to an AAV having capsid proteins fromserotype 1 and 5′-3′ ITRs from a different AAV serotype, e.g., AAVserotype 2.

The abbreviation “rAAV” refers to recombinant adeno-associated virus,also referred to as a recombinant AAV vector (or “rAAV vector”). In oneembodiment, the AAV expression vectors are constructed using knowntechniques to at least provide as operatively linked components in thedirection of transcription, control elements including a transcriptionalinitiation region, the DNA of interest and a transcriptional terminationregion. The control elements are selected to be functional in amammalian cell. The resulting construct which contains the operativelylinked components is flanked (5′ and 3′) with functional AAV ITRsequences.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus.

The nucleotide sequences of AAV ITR regions are known. As used herein,an “AAV ITR” need not have the wild-type nucleotide sequence depicted,but may be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV7, etc. Furthermore, 5′ and 3′ ITRs which flanka selected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a host cell genome or vector.

AAV ITRs can be excised from an AAV vector plasmid containing the sameand fused 5′ and 3′ of a selected nucleic acid construct that is presentin another vector using standard ligation techniques, such as thosedescribed in Sambrook and Russell, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press Cold Spring Harbor, N.Y.(2001). For example, ligations can be accomplished in 20 mM Tris-Cl pH7.5, 10 mM MgCl2, 10 mM DTT, 33 μg/ml BSA, 10 mM-50 mM NaCl, and either40 μM ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at 0° C. (for “stickyend” ligation) or 1 mM ATP, 0.3-0.6 (Weiss) units T4 DNA ligase at 14°C. (for “blunt end” ligation). Intermolecular “sticky end” ligations areusually performed at 30-100 μg/ml total DNA concentrations (5-100 nMtotal end concentration). AAV vectors which contain ITRs have beendescribed in, e.g., U.S. Pat. No. 5,139,941. In particular, several AAVvectors are described therein which are available from the American TypeCulture Collection (“ATCC”) under Accession Numbers 53222, 53223, 53224,53225 and 53226.

The adeno-associated virus preferentially packages a full-length genome,i.e., one that is approximately the same size as the native genome, andis not too big or too small. Many target nucleic acid sequences, orexpression cassettes encoding target nucleic acid sequences, are verysmall. To avoid packaging of fragmented genomes, the present inventorsdesigned and tested a nucleic acid sequence when linked to an expressioncassette, resulted in a genome whose size was near-normal in lengthbetween the ITRs. The starting sequence was of mammalian origin, but wassignificantly modified to ensure that this “filler component” (alsocalled a “stuffer sequence”) was devoid of enhancers, promoters,splicing regulators, noncoding RNAs or antisense sequences, among otherthings. In other words, the stuffer sequences are “silent” and confer noactivity to the expression cassette.

In the present invention, suitable DNA molecules for use in AAV vectorswill include, for example, a stuffer sequence and an expression cassetteencoding a siRNA molecule of the invention. Many expression cassettesare very small, for example, those expressing inhibitory RNAs (siRNAsand shRNAs). Thus, there is a need to add sequences to the cassette suchthat it makes up a full-length or near full-length AAV genome. If onlythe small genome was used in the AAV production, the recombinant virionswould be heterogeneous and contain various size genomes. This is becausethe virus likes to package full length genomes so it would pick up otherDNA fragments to fill that space. The stuffer cannot be too big, as AAVgenomes above 105% of the wild-type genome size will generally not bepackaged.

In certain embodiments, the present invention provides anadeno-associated virus (AAV) filler component (also called a “stuffersequence”) comprising a nucleic acid of between 3300 and 4200nucleotides in length having at least 90% identity to SEQ ID NO:1 or SEQID NO:2.

(SEQ ID NO: 1)GAATTCGGGCTATCCCAGGTTGCCTTGGTTCATGGCAAATGGGACGTTAAGAGGGCAGAGAGAATATGAACAGAAACTGTTCTAATATTGGTCATTTAATGTGTAAGTATTGTTCTTTTTTAAACCTCCTTCATTTTTTTTCCAGGAATTGCTGGACACAGTGGCTTGGTGTGTGTCTGAGGACTGTAGGCCATGGCCCTAGGTTGTGGTTTTAGGTCTCAGGTGCTCTTCCTGGCTGTCTCCTTGCTTCTTTCCCATGTCCTCTTCTTTGTTTCCAGCCATTTCTCCCTTATGCTTAAGTTTGGTGCAGCAGGGTTTGGCTGCTCTCAGATTCCTGCTTCCTCAGATGCTGTAGTTGTCAGGCCCAGCGGGCTGGCAGCGGGATCAGGATCTGGCTAGGTTTGCTCTCACTGTGGCAGAGTAGGGGGAGGCGTGGGAGAGCACGTGTGACCCCAGGCCAGCTGTAGGGAGCATAGGCATGGTCACGTAGCCTTCAGGTCCTAGACTTTGTCTTCTCATGAGTATGGCTGTGTGTGTATGGTGAAAACTAGGTTCTACTTAGCCCAAGAAAATGGGCACATTTTGCATGTGGTTTCTGTAGAGAAATGCACTGGGTATCTGACATAGCCTGGCAGCATGCCTCCCTCAGGTAGGTTAGTCTCAGGCGGTGAAGCACGTGTGTCCAGCAAGAACTTCATATGTGGCATAAAGTCTCCGTTCTGTGAGGTGCTGGCAAATCACCACCACCGTCAAGAGGCTGAAGTGATTTTTGTCTAGGGAGGCAGGAAAGGCTTCCTGGAGTCAGCAGCCAGTAGGTGAAAGAGTAGATTGGAGACCTTCTTAATCATCACCGCCTCTTGTCTCAAGGGGTGCCAGGAAGCTGTGGAGGCTGAACCCATCTTATGCTGCCAGAGAGTGGGACACCATGAGGGTCAGGTCAAGGGGTTGTACCTTGTTTGGTAGAGAATTAGGGGCTCTTGAAGACTTTGGATGTGGTCAGGGGAGTGTATCATTTAGGAAGAGTGACCCGGTGAGGACGTGGGGTAGAGGAGGACAGGTGGGAGGGAGTCCAGGTGGGAGTGAGTAGACCCAGCAGGAGTGCAGGGCCTCGAGCCAGGATGGTGGCAGGGCTGTGAGGAGAGGCAGCCACCTGTGTGTCTGCGGAAGCAGGGGCAAGAGGGAAGAGGCCAGCAGCGTGCTGCCATCACCCAGCGACTGGCGTAGATTGTGAGAGACCATTCCCTGCTCTTAGGAGGGGCTGAGTTTTAGTTTTCTCTTGTTATACAATAAGCTTGGTATTTGTTTACAAAACATTTGTAAAGCTAAATCAAGGTTTGATAAGGCTTCTAGTTTTATTTAAGAAGTAATGTTGAAATAAATGTTTGTCCAATTCGCTTTGCTCATTTAAGGACTTTCAGTACAAACTGCAACAACAGGATTAGGATTTAAACGTTTCTGAGATGTTTTTACTCCTCAGAATTTCCCAGAATGTGATCTGGTTTTGATTTTCAAGCTTGCTGACCCAATAGGTTAACCCACAAGTTTTACGAAGACCATCTCAGTCCACTTACATCAACTGCCCATGCCACGGTTAAAGAGATCATCGACTGATGTTTGGCACAGCTTCCTCCCTCTTGGGTGGGCAAGCATTTGGAAGAGAAGGCTCCTATGGGTGAGAGTGGGGCACCAAAGTCTTCCCTGTCCCATCCCCTAGCTTGAGAAGCCCTTCTCTAATGTGGACTTTGTGCCGTTAGCATCGTTACTAGCTTGAAGTTGACCATCTGGACGTACTTTCTGGTTTAGCCTCACAAGTGAGCAAGGAGGGTTGAGAGATGTGCTGTGAGGAATGTGGGGCCCCAGCTGGCAGCAGGCTCTGGGTCAGGGGGGCAGGGACCACGGGCATACCTGACAGTGAGGAGGGTCTAGTAGGGGATCAGTTCCCCTGTTGTTCTTTAGAATTTTCTGGATATTCTTCTTTATTGATTTTGGGATGTGAACAATAGAATCAACTTCTACTTGTAGATTGATTTAGGGAGAACTTATACCTCAGATGTTAAGTCACCCTGTCCAGAATGTGGGATGCTTTCCTATTTGTTCAGAACTTTTTAAATTACCTCAGAAGCACATGAAATTTAAAGGATTTTAAAAAAAACTTAAAGATTATTTCACATAGCTCTTGCACATTTCTTGATAAATGAATCCTCAGGTATTCCTCTGTTTTTGTTACTAATAGTTACTTCTTATGGGTTTTTTTTCCCCTGAAAATCATTTATCAAACGTATGTGGCTTATTTTCTGAAGGATGTTTGATAATTTTGGAAGATATGAAAGTCTTCATATTTTACAAGGTTTGAGGTCTCTTTAAGCTGCATGGTTCTCATGTCAGCTCCCAAAGCAGAAGACGGCATGTTGAAAAATGCCGTAGAGAAGATACTTCTTTTCCACCTGTTTTCAACTCATATCATCTTGAATTTCAGGGCACCTTTCCATGCTCCTAGTGCTTGCTATCTGTTTATTATTTTCCTTCCTGAATACCCTGAACTCCAGCATGTTCTGCTGTAATTCTGGCCTCCCTGGCATCTTGGACTCCTGTTTCCTTTGCTCTGTCATCCCCGCGGTCAGCTCCTGCTGCGCAGCTTCTCAGCTGAAGTGCGTTTGGAGTGCCTGGCGTGTCTTGCTGGATCTTTGAGTATTGCCTCTGGTTTCCTTGGTTCCTTCTGCTGAGTTGCTCAGCGTCTCCACTCCCCATTTCTTGTGTGGCCCTTCCTGCACTCCTCTGATTCCTTTTGTCTTCCCTGGTTTCTTGCTTTGGTTTCGAGTCTCCACAGAACTTTTGCAGCTCTTCTGAAGACCTGGAAGCTTTTTCATCTTAATTCTCATCTCATGACCTCTTTTCCCTTCTTTGAGAGCTAGAACTTCCCATGGTGAACTTCTCTTTCCAGAATTCCATGCCTTCTTTTCCCTCCCACTTACCTGTTGTCCAGGAGAGGTCAGATTGCTGTGCATATTGGAGGAGAACCCTTTCTTCCCTGGGCTCTTCATCTCACATGACATCACCACATCACCTCGTTCCTTGGACCCTCAGTGGTGTCACTGCTGGATTTTTCTTTCCTTTGGCTGGCCTTAGGGCACACCCAGGTTGACTAGCGTAGTCATGGTATTTAGATCCACTCACATTTTCAGTTTCTGTGTCTGTCTCTTGCCTGCTTCTGACTTCGCCCAGAGAAAGCTTCTCTTTCACAAGGGTTCTTAGATTTATGTTCACTGAGCACCTTCTTTTCTGAGGCAGTGTTTTACCAATATTTATTTTCCTAGTCAGTCTCGCCTTACCTTTCTTGTTATGCATGTCTTTGGTCCTGACCCATTCTCTGAGTCTGTAAAATAGAATTGCTGTATAATTTAATTACATGAAATCCTTTAGAATCTTAACACATCTTACACCTGATTTAATATTTTATTGTATCCAAATTGAACCAACCCTATGTGAATTTGACAGTGATTTCTCCCAGGGATCCTAGTGTATAAGGAATAGGACTTAGTATTTTCTATTTTTTGATATACCACATACCAGATACTGATTATGATGGACATTTAACCCTTTTTTCTCATTATGAAAGAAAGTTAGGAATTATTTCTTCCAGTAGCGCCAGTGTAACCTGAAAGCCTTTGAAAGAGTAGTTTTTGTATAGCTATCTGAAAGGAATTTCTTTCCAAAATATTTTTCCAGTGCTGACAACAAACACGCAGACACACCCTGCAAGGTGAGTGTACGGCG(SEQ ID NO: 2)GGGCTATCCCAGGTTGCCTTGGTTCATGGCAAATGGGACGTTAAGAGGGCAGAGAGAATATGAACAGAAACTGTTCTAATATTGGTCATTTAATGTGTAAGTATTGTTCTTTTTTAAACCTCCTTCATTTTTTTTCCAGGAATTGCTGGACACAGTGGCTTGGTGTGTGTCTGAGGACTGTAGGCCATGGCCCTAGGTTGTGGTTTTAGGTCTCAGGTGCTCTTCCTGGCTGTCTCCTTGCTTCTTTCCCATGTCCTCTTCTTTGTTTCCAGCCATTTCTCCCTTATGCTTAAGTTTGGTGCAGCAGGGTTTGGCTGCTCTCAGATTCCTGCTTCCTCAGATGCTGTAGTTGTCAGGCCCAGCGGGCTGGCAGCGGGATCAGGATCTGGCTAGGTTTGCTCTCACTGTGGCAGAGTAGGGGGAGGCGTGGGAGAGCACGTGTGACCCCAGGCCAGCTGTAGGGAGCATAGGCATGGTCACGTAGCCTTCAGGTCCTAGACTTTGTCTTCTCATGAGTATGGCTGTGTGTGTATGGTGAAAACTAGGTTCTACTTAGCCCAAGAAAATGGGCACATTTTGCATGTGGTTTCTGTAGAGAAATGCACTGGGTATCTGACATAGCCTGGCAGCATGCCTCCCTCAGGTAGGTTAGTCTCAGGCGGTGAAGCACGTGTGTCCAGCAAGAACTTCATATGTGGCATAAAGTCTCCGTTCTGTGAGGTGCTGGCAAATCACCACCACCGTCAAGAGGCTGAAGTGATTTTTGTCTAGGGAGGCAGGAAAGGCTTCCTGGAGTCAGCAGCCAGTAGGTGAAAGAGTAGATTGGAGACCTTCTTAATCATCACCGCCTCTTGTCTCAAGGGGTGCCAGGAAGCTGTGGAGGCTGAACCCATCTTATGCTGCCAGAGAGTGGGACACCATGAGGGTCAGGTCAAGGGGTTGTACCTTGTTTGGTAGAGAATTAGGGGCTCTTGAAGACTTTGGATGTGGTCAGGGGAGTGTATCATTTAGGAAGAGTGACCCGGTGAGGACGTGGGGTAGAGGAGGACAGGTGGGAGGGAGTCCAGGTGGGAGTGAGTAGACCCAGCAGGAGTGCAGGGCCTCGAGCCAGGATGGTGGCAGGGCTGTGAGGAGAGGCAGCCACCTGTGTGTCTGCGGAAGCAGGGGCAAGAGGGAAGAGGCCAGCAGCGTGCTGCCATCACCCAGCGACTGGCGTAGATTGTGAGAGACCATTCCCTGCTCTTAGGAGGGGCTGAGTTTTAGTTTTCTCTTGTTATACAATAAGCTTGGTATTTGTTTACAAAACATTTGTAAAGCTAAATCAAGGTTTGATAAGGCTTCTAGTTTTATTTAAGAAGTAATGTTGAAATAAATGTTTGTCCAATTCGCTTTGCTCATTTAAGGACTTTCAGTACAAACTGCAACAACAGGATTAGGATTTAAACGTTTCTGAGATGTTTTTACTCCTCAGAATTTCCCAGAATGTGATCTGGTTTTGATTTTCAAGCTTGCTGACCCAATAGGTTAACCCACAAGTTTTACGAAGACCATCTCAGTCCACTTACATCAACTGCCCATGCCACGGTTAAAGAGATCATCGACTGATGTTTGGCACAGCTTCCTCCCTCTTGGGTGGGCAAGCATTTGGAAGAGAAGGCTCCTATGGGTGAGAGTGGGGCACCAAAGTCTTCCCTGTCCCATCCCCTAGCTTGAGAAGCCCTTCTCTAATGTGGACTTTGTGCCGTTAGCATCGTTACTAGCTTGAAGTTGACCATCTGGACGTACTTTCTGGTTTAGCCTCACAAGTGAGCAAGGAGGGTTGAGAGATGTGCTGTGAGGAATGTGGGGCCCCAGCTGGCAGCAGGCTCTGGGTCAGGGGGGCAGGGACCACGGGCATACCTGACAGTGAGGAGGG G TCTAGTAGGGGATCAGTTCCCCTGTTGTTCTTTAGAATTTTCTGGATATTCTTCTTTATTGATTTTGGGATGTGAACAATAGAATCAACTTCTACTTGTAGATTGATTTAGGGAGAACTTATACCTCAGATGTTAAGTCACCCTGTCCAGAATGTGGGATGCTTTCCTATTTGTTCAGAACTTTTTAAATTACCTCAGAAGCACATGAAATTTAAAGGATTTTAAAAAAAACTTAAAGATTATTTCACATAGCTCTTGCACATTTCTTGATAAATGAATCCTCAGGTATTCCTCTGTTTTTGTTACTAATAGTTACTTCTTATGGGTTTTTTTTCCCCTGAAAATCATTTATCAAACGTATGTGGCTTATTTTCTGAAGGATGTTTGATAATTTTGGAAGATATGAAAGTCTTCATATTTTACAAGGTTTGGGGTCTCTTTAAGCTGCATGGTTCTCATGTCAGCTCCCAAAGCAGAAGACGGCATGTTGAAAAATGCCGTAGAGAAGATACTTCTTTTCCACCTGTTTTCAACTCATATCATCTTGAATTTCAGGGCACCTTTCCATGCTCCTAGTGCTTGCTATCTGTTTATTATTTTCCTTCCTGAATACCCTGAACTCCAGCATGTTCTGCTGTAATTCTGGCCTCCCTGGCATCTTGGACTCCTGTTTCCTTTGCTCTGTCATCCCCGCGGTCAGCTCCTGCTGCGCAGCTTCTCAGCTGAAGTGCGTTTGGAGTGCCTGGCGTGTCTTGCTGGATCTTTGAGTATTGCCTCTGGTTTCCTTGGTTCCTTCTGCTGAGTTGCTCAGCGTCTCCACTCCCCATTTCTTGTGTGGCCCTTCCTGCACTCCTCTGATTCCTTTTGTCTTCCCTGGTTTCTTGCTTTGGTTTCGAGTCTCCACAGAACTTTTGCAGCTCTTCTGAAGACCTGGAAGCTTTTTCATCTTAATTCTCATCTCATGACCTCTTTTCCCTTCTTTGAGAGCTAGAACTTCCCATGGTGAACTTCTCTTTCCAGAATTCCATGCCTTCTTTTCCCTCCCACTTACCTGTTGTCCAGGAGAGGTCAGATTGCTGTGCATATTGGAGGAGAACCCTTTCTTCCCTGGGCTCTTCATCTCACATGACATCACCACATCACCTCGTTCCTTGGACCCTCAGTGGTGTCACTGCTGGATTTTTCTTTCCTTTGGCTGGCCTTAGGGCACACCCAGGTTGACTAGCGTAGTCATGGTATTTAGATCCACTCACATTTTCAGTTTCTGTGTCTGTCTCTTGCCTGCTTCTGACTTCGCCCAGAGAAAGCTTCTCTTTCACAAGGGTTCTTAGATTTATGTTCACTGAGCACCTTCTTTTCTGAGGCAGTGTTTTACCAATATTTATTTTCCTAGTCAGTCTCGCCTTACCTTTCTTGTTATGCATGTCTTTGGTCCTGACCCATTCTCTGAGTCTGTAAAATAGAATTGCTGTATAATTTAATTACATGAAATCCTTTAGAATCTTAACACATCTTACACCTGATTTAATATTTTATTGTATCCAAATTGAACCAACCCTATGTGAATTTGACAGTGATTTCTCCCAGGGATCCTAGTGTATAAGGAATAGGACTTAGTATTTTCTATTTTTTGATATACCACATACCAGATACTGATTATGATGGACATTTAACCCTTTTTTCTCATTATGAAAGAAAGTTAGGAATTATTTCTTCCAGTAGCGCCAGTGTAACCTGAAAGCCTTTGAAAGAGTAGTTTTTGTATAGCTATCTGAAAGGAATTTCTTTCCAAAATATTTTTCCAGTGCTGACAACAAACACGCAGACACACCCTGCAAGGTGAGTGTACGGCG

In certain embodiments, the present invention provides anadeno-associated virus (AAV) filler component consisting of a nucleicacid of between 3300 and 4200 nucleotides in length having at least 90%identity to SEQ ID NO:1 or SEQ ID NO:2. In certain embodiments, thefiller component consists of at least 90% identity with SEQ ID NO:1 orSEQ ID NO:2. In certain embodiments, the filler component has 95%identity, 98% identity, 99% identity, or even 100% identity with SEQ IDNO:1 or SEQ ID NO:2. In certain embodiments, the filler component has alength of about 3500-4000 nucleotides, or of about 3700-3850nucleotides. In the present invention, the filler component is “silent”in terms of biological activity, in that it is devoid of enhancers,promoters, splicing regulators, noncoding RNAs, antisense sequences, orcoding sequences.

The term “nucleic acid” refers to deoxyribonucleic acid (DNA) orribonucleic acid (RNA) and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base that is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. A “nucleic acid fragment” is a portion of a givennucleic acid molecule.

A “nucleotide sequence” is a polymer of DNA or RNA that can besingle-stranded or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases capable of incorporation intoDNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,”“nucleic acid fragment,” “nucleic acid sequence or segment,” or“polynucleotide” are used interchangeably and may also be usedinterchangeably with gene, cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleicacid compositions. In the context of the present invention, an“isolated” or “purified” DNA molecule or RNA molecule is a DNA moleculeor RNA molecule that exists apart from its native environment and istherefore not a product of nature. An isolated DNA molecule or RNAmolecule may exist in a purified form or may exist in a non-nativeenvironment such as, for example, a transgenic host cell. For example,an “isolated” or “purified” nucleic acid molecule or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. In one embodiment, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Fragments andvariants of the disclosed nucleotide sequences are also encompassed bythe present invention. By “fragment” or “portion” is meant a full lengthor less than full length of the nucleotide sequence.

“Naturally occurring,” “native,” or “wild-type” is used to describe anobject that can be found in nature as distinct from being artificiallyproduced. For example, a protein or nucleotide sequence present in anorganism (including a virus), which can be isolated from a source innature and that has not been intentionally modified by a person in thelaboratory, is naturally occurring.

“Genome” refers to the complete genetic material of an organism.

A “vector” is defined to include, inter alia, any viral vector, as wellas any plasmid, cosmid, phage or binary vector in double or singlestranded linear or circular form that may or may not be selftransmissible or mobilizable, and that can transform prokaryotic oreukaryotic host.

AAV ITRs

An “AAV virus” or “AAV viral particle” refers to a viral particlecomposed of at least one AAV capsid protein (preferably by all of thecapsid proteins of a wild-type AAV) and an encapsidated polynucleotide.If the particle comprises heterologous polynucleotide (i.e., apolynucleotide other than a wild-type AAV genome such as a transgene tobe delivered to a mammalian cell), it is typically referred to as“rAAV”.

In one embodiment, the AAV expression vectors are constructed usingknown techniques to at least provide as operatively linked components inthe direction of transcription, control elements including atranscriptional initiation region, the DNA of interest and atranscriptional termination region. The control elements are selected tobe functional in a mammalian cell. The resulting construct whichcontains the operatively linked components is flanked (5′ and 3′) withfunctional AAV ITR sequences.

By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” ismeant the art-recognized regions found at each end of the AAV genomewhich function together in cis as origins of DNA replication and aspackaging signals for the virus. AAV ITRs, together with the AAV repcoding region, provide for the efficient excision from plasmidsexpressing them.

The nucleotide sequences of AAV ITR regions are known. As used herein,an “AAV ITR” need not have the wild-type nucleotide sequence depicted,but may be altered, e.g., by the insertion, deletion or substitution ofnucleotides. Additionally, the AAV ITR may be derived from any ofseveral AAV serotypes, including without limitation, AAV1, AAV2, AAV3,AAV4, AAV5, AAV7, etc. Furthermore, 5′ and 3′ ITRs which flank aselected nucleotide sequence in an AAV vector need not necessarily beidentical or derived from the same AAV serotype or isolate, so long asthey function as intended, i.e., to allow for excision and rescue of thesequence of interest from a vector, and to package the desired genomeinto the AAV virion.

In one embodiment, AAV ITRs can be derived from any of several AAVserotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5,AAV7, etc. Furthermore, 5′ and 3′ ITRs which flank a selected nucleotidesequence in an AAV expression vector need not necessarily be identicalor derived from the same AAV serotype or isolate, so long as theyfunction as intended, i.e., to allow for excision and rescue of thesequence of interest from a vector, and to allow packaging of thedesired genome into the AAV virion.

In certain embodiments, the present invention provides anadeno-associated virus (AAV) vector comprising the filler component asdescribed above operably linked to an expression cassette. In certainembodiments, the expression cassette comprises a promoter. In certainembodiments, the promoter is a pol III promoter. In certain embodiments,the promoter is a mU6 promoter. In certain embodiments, the AAV vectorfurther comprising a target sequence. In certain embodiments, the targetsequence is an RNAi molecule.

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, which may include a promoter operably linkedto the nucleotide sequence of interest that may be operably linked totermination signals. The coding region usually codes for a functionalRNA of interest, for example an RNAi molecule. The expression cassetteincluding the nucleotide sequence of interest may be chimeric. Theexpression cassette may also be one that is naturally occurring but hasbeen obtained in a recombinant form useful for heterologous expression.

Double-stranded RNA (dsRNA) can induce sequence-specificposttranscriptional gene silencing in many organisms by a process knownas RNA interference (RNAi). RNA fragments are the sequence-specificmediators of RNAi. Interference of gene expression by these RNAinterference (RNAi) molecules is now recognized as a naturally occurringstrategy for silencing genes in the cells of many organisms.

Certain embodiments of the present invention provide a vector thatencodes an isolated RNAi molecule. As used herein the term “encoded by”is used in a broad sense, similar to the term “comprising” in patentterminology. RNAi molecules include siRNAs, shRNAs and other small RNAsthat can or are capable of modulating the expression of a target gene,for example via RNA interference. Such small RNAs include withoutlimitation, shRNAs and miroRNAs (miRNAs).

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one of thesequences is affected by another. For example, a regulatory DNA sequenceis said to be “operably linked to” or “associated with” a DNA sequencethat codes for an RNA or a polypeptide if the two sequences are situatedsuch that the regulatory DNA sequence affects expression of the codingDNA sequence (i.e., that the coding sequence or functional RNA is underthe transcriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

Operably linked nucleic acids are nucleic acids placed in a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, operably linked DNA sequences are DNA sequencesthat are linked are contiguous. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

The invention will now be illustrated by the following non-limitingExamples.

Example 1

A plasmid FBAAVmU6miHDS1stuffer was generated that included AAV2 ITRs,mU6 promoter, miHDS1 target sequence, filler component stuffer, and anAAV backbone (FIG. 1). The sequence for 5pFBAAVmU6miHDS1AAVstuffer isprovided in FIGS. 2A-2R, and the sequences for the individual componentsof the plasmid are provided in FIGS. 3A-3C. The full-length fillercomponent (“stuffer sequence”) consisted of 3776 nucleotides.

Example 2

The in vivo silencing efficiency of a vectors expressing miHDS1 wascompared. Four vectors were constructed: (1) a vector expressing acontrol sequence (miSAFE) and containing a control sequence (eGFP), (2)a vector expressing the target sequence (miHDS1) and containing acontrol sequence (eGFP), (3) a vector expressing a control sequence(miSAFE) and containing the stuffer sequence described in Example 1, and(4) a vector expressing the target sequence (miHDS1) and containing thestuffer sequence described in Example 1.

(1) AAV2/1 mU6miSAFE—eGFP (4.81 E12 μg/ml)

(2) AAV2/1 mU6miHDS1—eGFP (4.81 E12 μg/ml)

(3) AAV2/1 mU6miSAFE—stuffer (4.81 E12 μg/ml)

(4) AAV2/1 mU6miHDS1—stuffer (4.81 E12 μg/ml)

The sequences for miSAFE and miHDS1 have been previously discussed (see,PCT/US2012/024904, which is hereby incorporated by reference herein inits entirety). Wild type mice were injected in the striatum with thefour vectors. Mice were sacrificed one month later and Htt expressionwas determined relative to Actb expression levels by QPCR. FIG. 4 showsthat there was a 20% decrease in expression between the misafe/eGFP andthe miHDS1/eGFP expression cassettes, whereas there was a 60% decreasein expression between the misafe/stuffer and the miHDS1/stufferexpression cassettes, i.e., a 60% decrease in expression when thestuffer was used.

Example 3

A plasmid 5pFBAAVmU6miHDS1stuffer was generated that included AAV2 ITRs,mU6 promoter, miHDS1 target sequence, filler component stuffer, and anAAV backbone (FIG. 5). The sequence for the plasmid5pFBAAVmU6miHDS1AAV-stuffer is provided in FIGS. 6A-6D. The sequence forthe stuffer (Stuffer #2) is provided in FIGS. 7A-7B.

Example 4

One of the considerations with AAV packaging is maintaining optimalgenome size. When this occurs, the ratio of virions that form which arelacking genomes are minimized. Experiments were performed testing thepackaging efficiency of the new stuffer sequences and found highefficiency packaging. For example, see Table 1 “Average empty” and FIG.8). It was also measured if genetic material that was packaged containednon-miRNA:intron stuffer sequences. It was found that the incorporationof unintended genomic material used in virus production was extremelylow (Cap/rAAV, Amp/rAAV, Gent/rAAV). Finally, the quality of the viruseswere analyzed by Silver Stain after polyacrylamide gel electrophoresisand found to contain the appropriate proportions of the various capsidproteins (VP1, VP2, and VP3; FIG. 9). In summary, the intron I/II stuffsequence allows optimal packaging of desired transgenes into AAVcapsids.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

What is claimed is:
 1. An adeno-associated virus (AAV) filler componentcomprising a nucleic acid of between 3300 and 4200 nucleotides in lengthhaving at least 90% identity to SEQ ID NO:1 or SEQ ID NO:2.
 2. Anadeno-associated virus (AAV) filler component consisting of a nucleicacid of between 3300 and 4200 nucleotides in length having at least 90%identity to SEQ ID NO:1 or SEQ ID NO:2.
 3. The AAV filler component ofclaim 1 or claim 2, wherein the nucleic acid is between 3500 and 4000nucleotides.
 4. The AAV filler component of claim 1 or claim 2, whereinthe nucleic acid is between 3700 and 3850 nucleotides.
 5. The AAV fillercomponent of any one of claims 1 to 4, wherein the nucleic acid has atleast 95% identity to SEQ ID NO:1 or SEQ ID NO:2.
 6. An adeno-associatedvirus (AAV) vector comprising the filler component of any one of claims1 to 5 operably linked to an expression cassette.
 7. The AAV vector ofclaim 6, wherein the expression cassette comprises a promoter.
 8. TheAAV vector of claim 7, wherein the promoter is a pol III promoter. 9.The AAV vector of claim 8, wherein the promoter is a mU6 promoter. 10.The AAV vector of any one of claims 6 to 9 further comprising a targetsequence.
 11. The AAV vector of claim 10, wherein the target sequence isan RNAi molecule.